During FY08, we focussed primarily on four subprojects.[unreadable] [unreadable] (1) Hepatitis B Virus Capsid Assembly. We study the HBV capsid protein which presents two of the three clinically important antigens - core antigen (capsids) and e-antigen (unassembled protein) - of this major human pathogen. After first showing that capsid protein self-assembles from dimers into capsids of two different sizes, we obtained, in 1997, a cryo-EM density map in which much of the secondary structure was visible, including the 4-helix bundle that forms the dimerization motif. This was the first time that such detailed information had been achieved by cryo-EM. Our subsequent research helped delineate the path of the polypeptide chain. We went on to investigate the antigenic diversity of HBV by using cryo-EM to characterize the conformational epitopes of seven different monoclonal antibodies raised against capsids. In FY08, we followed two lines of investigation. In one, we employed the technique of surface plasmon resonance to measure the binding affinities of a set of monoclonal antibodies commonly used to discriminate between core- and e-antigen, including several that we previously characterized by cryo-EM. Unexpectedly, almost all of the antibodies bind to both antigens with high affinity. The exceptions are antibody e6 which detects an epitope accessible only on dimers and occluded on capsids, and antibody 3120 which detects an epitope presented only on capsids because its epitope spans an inter-dimer interface. In the second project, "native" high resolution mass spectrometry was used to determine the masses of both size variants of the capsid to within 0.1%. It follows from these data that both lattices are complete, consisting of exactly 180 and 240 subunits. This experimental approach, anchored on very precise and accurate mass measurements, appears to hold considerable potential for elucidating the assembly of viruses and other macromolecular particles.[unreadable] [unreadable] (2) Structure, Assembly, and Maturation of Herpesviruses. Herpesviruses are large complex DNA viruses, eight members of which cause diseases in humans, including skin diseases such as Kaposi's sarcoma which is prevalent in immunosuppressed AIDS patients. In this long-term project, we first defined the molecular anatomy of the herpesvirus capsid and then characterized the roles of its six major proteins in assembly. In these properties, herpesviruses differ from other animal viruses but closely resemble tailed bacteriophages, suggesting a distant evolutionary relationship. One major finding was our discovery, with J. Brown, of the HSV procapsid, which differs radically from the mature capsid in structure and stability. Maturation of the procapsid, controlled by the viral protease, is a potential target for antiviral drugs. In 2007, we completed and and published a cryo-EM study of the capsid-binding properties of UL25 and UL17, two minor but essential capsid proteins implicated in DNA packaging and capsid maturation. We found that C-capsids (mature DNA-filled capsids) have an elongated molecule present at a unique vertex-adjacent site that is not seen on unfilled capsids, and adduced biochemical data indicaing that (i) the C-capsid-specific component is a heterodimer of UL25 and UL17; and (ii) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. Binding of the ordered population is facilitated by structural changes induced on the outer surface as a result of DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus. In FY08, we have been seeking to confirm this assignment by labelling C-capsids with Fab fragments specific for UL25 or UL17.[unreadable] [unreadable] (3) Assembly and Maturation of Bacteriophage Capsids. Our interest in capsid assembly lies in the massive conformational changes that accompany their maturation. These transitions afford unique insights into allosteric regulation. We study maturation of several phages to exploit expedient aspects of each system. The tailed phages afford an excellent model for herpesvirus capsids (see (2) above), reflecting common evolutionary origins. In FY08, we focussed on two projects. [unreadable] (i) We used a combination of scanning calorimetry and cryo-EM to investigate the encapsidation of phage DNA, which represents an extreme case of genome condensation. Phage HK97 is well suited to study this phenomenon in view of detailed knowledge of its capsid structure. We found that, as filled capsids are heated, their DNA is released at relatively low temperatures (40 to 50 degrees). Heating increases the internal pressure, causing the capsid to rupture, releasing the DNA. DNA packaging also induces a change in the capsid structure that is reflected both in an earlier onset of thermal denaturation than empty capsids and in subtle morphological differences. (We also documented a similar effect in herpesvirus capsids - see (2) above). We envisage that this transition in the capsid shell is transmitted to the portal, altering its interactions with the packaging enzyme and thus signaling that packaging is complete. [unreadable] (ii) We completed the initial phase of a project to investigate capsid maturation of phage phi6, which has a tripartite genome of double-stranded RNA. It has been hypothesized that the phi6 procapsid matures in stepwise fashion, coupled to sequential packaging of the three segments. The RNA-dependent RNA polymerase (RdRP) of phi6 functions in the capsid interior in both replication and transcription. The procapsid is composed of the P1 protein forming the main shell, and three other proteins, including P2, the RdRP, which is incorporated into the assembling procapsid. To determine its location, we performed cryo-EM of wild type and several mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the three-fold axes that are lacking in a P2-null mutant; faint in wild-type reflecting the copy number of 3; and strong in a mutant with 10 copies of P2 per particle. We infer that, during maturation, the RdRP molecules rotate to positions close to adjacent five-fold vertices where they participate in replication and transcription. [unreadable] [unreadable] (4) Papillomaviruses and polyomaviruses. (i) In FY08, we started a project to analyze the structure of encapsidated SV40 chromosomes by cryo-electron tomography. Its primary goals are to visualize, quantitate, and determine the arrangement of nucleosomes; and to investigate contacts between the chromatin and the inner surface of the capsid. In a set of 100 reconstructed virions, we find them to contain a variable number, 19 +/-2 nucleosome-sized densities, whose distribution does not follow the icosahedral symmetry of the capsid shell. The tomograms also suggest the existence of specific contacts between the capsid shell and the enclosed chromatin.[unreadable] (ii) The major papillomavirus capsid protein L1 forms a 72-pentamer icosahedral shell. Although the minor capsid protein L2 is not required for capsid formation, it is thought to participate in genome encapsidation and other roles in the infectious entry pathway. In FY08, we completed a project to determine the abundance of L2 and its arrangement within the virion. Biochemical analysis showed that up to 72 molecules of L2 can be incorporated per capsid. Cryo-EM a revealed an L2-specific density beneath the axial lumen of each L1 pentamer. This structural information should facilitate investigation of L2 function during the papillomavirus life cycle.